Aromatase induction in tamoxifen-resistant breast cancer: Role of phosphoinositide...

1

3

4

5

6

7 Q1

89

10 Q211

12

1 4

1516171819

202122232425

2 6

Q3

45

46

47

48

49

50

51

52

53

54

55

56

Cancer Letters xxx (2014) xxx–xxx

CAN 11883 No. of Pages 9, Model 5G

19 May 2014

Contents lists available at ScienceDirect

Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Aromatase induction in tamoxifen-resistant breast cancer: Roleof phosphoinositide 3-kinase-dependent CREB activation

http://dx.doi.org/10.1016/j.canlet.2014.05.0030304-3835/� 2014 Published by Elsevier Ireland Ltd.

Abbreviations: ER, estrogen receptor; PI3K, phosphoinositide 3-kinase; CREB,cAMP response element binding protein; ERK, extracellular signal-regulated kinase;GR, glucocorticoid receptor; PTEN, phosphatase and tensin homolog; PGE2,phostaglandin E2; TAM, tamoxifen; COX-2, cyclooxygenase-2; EP1, PGE2 receptor1; EP2, PGE2 receptor 2; 5-Aza, 5-aza-20-deoxycytidine; MAPK, mitogen-activatedprotein kinase.⇑ Corresponding author. Tel.: +82 2 880 7851; fax: +82 2 872 1795.

E-mail address: [email protected] (K.W. Kang).

Please cite this article in press as: N.T.T. Phuong et al., Aromatase induction in tamoxifen-resistant breast cancer: Role of phosphoinositide 3-kinase-depCREB activation, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.003

Nguyen Thi Thuy Phuong a,d, Sung Chul Lim b, Young Mi Kim c, Keon Wook Kang d,⇑a College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Koreab Department of Pathology, College of Medicine, Chosun University, Gwangju 501-759, Republic of Koreac College of Pharmacy, Hanyang University, Republic of Koread College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea

2728293031323334353637

a r t i c l e i n f o

Article history:Received 28 December 2013Received in revised form 28 April 2014Accepted 1 May 2014Available online xxxx

Keywords:Tamoxifen resistancePI3K/AktAromataseCREB

3839404142

a b s t r a c t

Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast can-cer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can belimited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estro-gens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basalexpression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant humanbreast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromataseimmunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared tonon-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase(ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase(PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, wehave also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activationwas required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest thataromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREBactivation.

� 2014 Published by Elsevier Ireland Ltd.

43

44

57

58

59

60

61

62

63

64

65

66

67

68

Introduction

Breast cancer is one of the most common malignancies world-wide and estrogen is the primary hormone stimulant in the uncon-trolled growth of estrogen receptor (ER)-positive breast cancer[1,2]. A clinical study revealed that the concentration of estrogenin breast tumor tissues is several-fold higher than that in theplasma of postmenopausal breast cancer patients [3] and most ofthese patients show a resistance to tamoxifen (TAM), a representa-tive ER antagonist [4,5]. Aromatase is a member of cytochromeP450 superfamily important for sexual development, whose func-tion is to aromatize androgens to produce estrogens. In humans,

69

70

71

72

73

74

75

76

the gene CYP19, located on chromosome 15q21.1, encodes the aro-matase enzyme [6], and circulating C19 steroid precursors areessential substrates for extragonadal estrogen synthesis [7]. Aro-matase inhibitors have become useful for the management of ER-positive breast cancer patients [8–10].

Numerous studies have described the regulation of aromataseexpression and its relevance in breast cancer development. Gener-ally, aromatase is expressed at a higher level in human breast can-cer tissues than in normal breast tissues [11,12], and aromataseexpression is specifically regulated through the alternative use ofexon 1 and multiple promoters [13,14]. In addition, growth factorssecreted by breast cancer cells stimulate aromatase expression inboth breast cancer and adjacent adipose tissues [15].

Despite an initial response to TAM, the majority of ER-positivepatients will frequently relapse. Hence, TAM resistance is a majorchallenge in the management of breast cancer patients [4]. Aroma-tase inhibitors such as letrozole and exemestane are well tolerated,more effective than TAM in postmenopausal breast cancer patients[16], and considered as a therapeutic option for TAM-resistantbreast cancer. Several clinical trials have revealed a significant

endent

http://dx.doi.org/10.1016/j.canlet.2014.05.003

mailto:[email protected]


http://www.sciencedirect.com/science/journal/03043835

http://www.elsevier.com/locate/canlet


77

78

79

80

81

82

83

84

85

86

87

888990919293949596979899

100101102103104105

106

107108109110111112113

114

115116117118119120121122123124125

126

127128129130131132133134135136

137

138139140141142

143144145146

147

148149150151152153154155156

157

158159160161162163164165166167168169170

171

172173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

2 N.T.T. Phuong et al. / Cancer Letters xxx (2014) xxx–xxx


19 May 2014

benefit in sequential treatment with an aromatase inhibitor afterTAM administration over TAM-only therapy [17,18]. However,the regulation of aromatase expression regulation in TAM-resistantbreast cancer is poorly understood. In this study, we demonstratefor the first time that expression and activity of aromatase inTAM-resistant human breast cancer (TAMR-MCF-7) cells areincreased compared to control MCF-7 cells. And, we further triedto find out the signaling pathway(s) mediating aromatase up-reg-ulation in TAMR-MCF-7 cells.

Materials and methods

Materials

Antibodies against aromatase were purchased from Abcam (Cambridge, UK,Cat#: ab18995). Antibodies recognizing CREB, P-CREB, P-p38 kinase, p38 kinase,P-extracellular signal-regulated kinase (ERK) (p-ERK T202/Y204), ERK, p-Akt (p-Akt S473) and Akt were obtained from Cell Signaling Technology (Beverly, MA).Antibodies against phosphatase and tensin homolog (PTEN) and glucocorticoidreceptor (GR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Horseradish peroxidase-conjugated donkey anti-rabbit, anti-goat IgG, and alkalinephosphatase-conjugated donkey anti-mouse IgG were acquired from JacksonImmunoresearch Laboratories (West Grove, PA). Anti-actin antibody and most ofthe reagents used for molecular studies were obtained from Sigma (St. Louis,MO). pGRE-Luc plasmid and pcDNA-PTEN, a PTEN overexpressing vector weredonated from Dr. Lee KY (Chonnam National University, Gwangju, South Korea).pCRE-Luc was purchased from Stratagene (La Jolla, CA, USA). The aromatase-Lucreporter plasmid was kindly provided from Dr. Jeong HG (Chungnam National Uni-versity, Daejeon, South Korea). Myc-p85 (a dominant negative form of PI3K) andp110-myc (constitutive active form of PI3K) overexpression vectors were providedby Dr. A. Toker (The Boston Biomedical Research Institute, Boston, MA) and Dr. J.Downward (Imperial Cancer Research Fund, London) [19].

Cell culture and establishment of TAMR-MCF-7 cell

MCF-7, Raw 264.7 (mouse monocyte cell line) and mouse embryonic fibroblast(MEF) cells were cultured at 37 �C in 5% CO2/95% air in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% fetal bovine serum, 100 units/ml penicillin, and100 lg/ml streptomycin. Hormone-dependent T47D:A18/Neo cells and hormone-independent T47D:A18/PKCa cells were cultured in RPMI-1640 medium containing10% FBS, 100 units/ml penicillin, and 100 lg/ml streptomycin. TAMR-MCF-7 cellswere established as previously reported [20–22].

Immunoblot analysis

After washing with sterile PBS, cells were lysed in lysis buffer containing 20 mMTris–Cl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mMEDTA, 1 mM sodium orthovanadate, 25 mM b-glycerolphosphate, 2 mM sodiuminorganic pyrophosphate, 1 mM phenylmethylsulfonylfluoride, and 1 lg/ml leu-peptin. Total cell lysates were centrifuged at 10,000g for 10 min to remove cell deb-ris, and proteins in the supernatant were fractionated using a 10% separating gel.The fractionated proteins were then transferred electrophoretically to nitrocellu-lose paper, and the proteins were immunoblotted with specific antibodies. Anti-body dilution ratio used for western blotting was 1:1000. Protein concentrationin samples was measured by using Protein measurement kit (iNtron biotechnology,Seongnam, Korea).

Preparation of nuclear extracts

Cells in the dishes were washed with ice-cold PBS. The cells were then scraped,transferred to microtubes, and allowed to swell after adding 100 ll of a hypotonicbuffer containing 10 mM HEPES (pH 7.9), 10 mMKCl, 0.1mMEDTA, 0.5% Nonidet P-40, 1mMdithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride. The lysates wereincubated for 10 min on ice and centrifuged at 7200g for 5 min at 4 �C. Pellets con-taining the crude nuclei were resuspended in 50 ll of an extraction buffer contain-ing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, and1 mM phenylmethylsulfonylfluoride and incubated for 30 min on ice. The sampleswere centrifuged at 15,800g for 10 min to obtain supernatants containing thenuclear fractions. The nuclear fractions were stored at �80 �C until needed.

Reporter gene analysis

A dual-luciferase reporter gene assay system (Promega, Madison, WI) was usedto determine promoter activity. Briefly, cells were plated in 12-well plates and tran-siently transfected with 1 lg/ml reporter plasmids and phRL-SV plasmid (hRenillaluciferase expression for normalization) using Hillymax� reagent (Dojindo Molecu-lar Technologies, Gaithersburg, MD). The cells were then incubated in culture med-

Please cite this article in press as: N.T.T. Phuong et al., Aromatase induction in tamCREB activation, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.0

ium without serum for 18 h. Firefly and hRenilla luciferase activities in the celllysates were measured using a luminometer (LB941, Berthold Technologies, BadWild, Germany). Relative luciferase activities were calculated by normalizing thepromoter-driven firefly luciferase activity to the hRenilla luciferase.

Determination of aromatase activity

Aromatase activity was measured in both MCF-7 and TAMR-MCF-7 cells with atritiated water release assay [23]. 80% confluent MCF-7 and TAMR-MCF-7 cells wereincubated in serum-free medium for 36 h and the cells were treated with [1b-3H]androst-4-ene-3,17-dione (100 nM) for an additional 3 h. The medium was thenmixed thoroughly with 5% charcoal/0.5% dextran for 12 h and centrifuged at10,000g for 30 min at 4 �C to remove any residual androst-4-ene-3,17-dione. Oneml of the supernatants were added in a scintillation vial containing 3 ml scintilla-tion cocktail, and radioactivity was measured as disintegrations per minute usinga liquid scintillation counter (LS 6500, Beckman Coulter Inc., Fullerton, CA).

Immunohistochemistry for human cancer tissues

Blocks for all the samples were consecutively cut in 4 lm sections and mountedon poly-l-lysine coated glass slides. Xylene was used to remove the paraffin fromthe sections, and the samples were rehydrated. Antigen retrieval was performedby boiling sections for 5 min in 1 lM sodium citrate buffer (pH 6.0) in a microwaveoven. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide inmethanol for 10 min, followed by three times washing with PBS. Sections were thenincubated overnight with anti-aromatase antibody (1:200, Abcam, Cambridge, UK)at 4 �C. After washing with PBS, sections were incubated with HRP-conjugated anti-rabbit IgG for 30 min and washed with PBS. The color was developed by incubationwith DAB solution. Finally, sections were counterstained with hematoxylin, dehy-drated, mounted, and observed. When we determined immunoreactivity in rabbitIgG-incubated breast cancer tissue samples (negative control), we could not detectany positive staining.

Statistical analysis

Student’s t test was used to examine between group differences. Statistical sig-nificance was accepted at either P < 0.05 or P < 0.01.

Results

Up-regulation of aromatase expression in TAM-resistant human breastcancer

As aromatase activity plays an important role in breast cancerdevelopment through estrogen synthesis [11], we comparedchanges in aromatase protein expression and activity in MCF-7and TAMR-MCF-7 cells. Western blotting revealed that the proteinexpression of aromatase was significantly increased in TAMR-MCF-7 cells compared to MCF-7 cells (Fig. 1A). Reporter gene analysisusing an aromatase-luciferase (luc) reporter plasmid containingthe �294/+20 bp promoter region of rat aromatase gene [24]showed that reporter activity was higher in TAMR-MCF-7 cellsthan in MCF-7 cells (Fig. 1B). We also noted that basal aromataseactivity was consistently enhanced in TAM-MCF-7 cells (Fig. 1C).To confirm these results in human cancer cases, tumor tissues wereobtained from two groups of patients, which differed in terms ofthe occurrence of relapse after TAM therapy. Four cases includedin ‘‘Non-recurrence group after TAM therapy’’ experienced norecurrence for at least 6 years of follow-up after mastectomy withadjuvant TAM therapy. The other four cases in ‘‘Recurrence groupafter TAM therapy’’ relapsed within 3–4 years after mastectomywith adjuvant TAM therapy. Immunohistochemical analysesshowed that aromatase-positive cell number was significantlyhigher in recurrence group after TAM therapy than in non-recur-rence group after TAM therapy (Fig. 1D and E). To confirm theresults and aromatase band size, we determined the basal levelof aromatase expression in diverse cell types, including Raw264.7cells that have no expression of aromatase (negative control),and MEF cells where aromatase expression can be detected bywestern blot [25], and another tamoxifen resistant breast cancercell line T47D:A18/PKCa that is stably overexpressing PKCa and

oxifen-resistant breast cancer: Role of phosphoinositide 3-kinase-dependent03


Fig. 1. Up-regulation of aromatase expression in TAMR-MCF-7 cells. (A) Immunoblot analysis of aromatase in MCF-7 and TAMR-MCF-7 cells. Each lane represents differentsample. (B) Basal aromatase-luc activities in MCF-7 and TAMR-MCF-7 cells. MCF-7 and TAMR-MCF-7 cells were transiently transfected with aromatase-luc reporter (1 lg/ml)and phRL-SV (hRenilla) (1 ng/ml) plasmids. Dual luciferase reporter assays were performed on the lysed cells 18 h after transfection. The transfected cells were incubated inserum-free medium. Reporter gene activity was calculated as a relative ratio of firefly luciferase to hRenilla luciferase activity. Data represent mean ± SD with 6 differentsamples (significant versus MCF-7 cells, ��P < 0.01). (C) Increase in aromatase activity in TAMR-MCF-7 cells compared to MCF-7 cells. Data represent mean ± SD with 3different samples (significant versus MCF-7 cells, ��P < 0.01). (D), (E) representative figures for aromatase immunohistochemistry in human breast cancer tissues of 2 groupsof patients: (D) Non-recurrence group after TAM therapy; (E) Recurrence group after TAM therapy, respectively. Positive stained cell number was described (n = 4) (significantversus non-recurrence group after TAM therapy, ��P < 0.01). Aromatase-positive stained cell number was counted in three different fields of each tissue sample, and averageand standard deviation values were described. (F) Expression of aromatase in diverse cell types: Raw264.7 (negative control); estrogen-dependent breast cancer cell line,T47D: A18/Neo; estrogen-independent breast cancer cell line, T47D:A18/PKCa and MEF cells (positive control).

N.T.T. Phuong et al. / Cancer Letters xxx (2014) xxx–xxx 3


19 May 2014

Please cite this article in press as: N.T.T. Phuong et al., Aromatase induction in tamoxifen-resistant breast cancer: Role of phosphoinositide 3-kinase-dependentCREB activation, Cancer Lett. (2014), http://dx.doi.org/10.1016/j.canlet.2014.05.003


205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240



19 May 2014

ER function is down-regulated [26]. The aromatase band size inTAMR-MCF-7 cells and MEF cells was exactly the same (Fig. 1F).Moreover, aromatase expression was also enhanced in T47D:A18/PKCa cells compared to T47D: A18 cells, suggesting that aromataseis activated in TAM-resistant breast cancer.

Involvement of CREB activation in the up-regulation of aromataseexpression in TAMR-MCF-7 cells

To better understand the regulation of aromatase expression inTAMR-MCF-7 cells, we further examined the transcription factorsrequired for aromatase induction. Several studies have shown thatCREB binding to the cAMP response element (CRE) or glucocorti-coids binding to the glucocorticoid response element (GRE) in thepromoter region of the aromatase gene regulates transcription ofaromatase in breast cancer cells [27–29]. When we assessed theminimal reporter activities of CRE and GRE in both cell types, thebasal CRE-luc reporter activity was distinctly higher inTAMR-MCF-7 cells than in MCF-7 cells (Fig. 2A). However, GRE-luc reporter activity was not significantly different between the

Fig. 2. Role of CREB in the up-regulation of aromatase expression in TAMR-MCF-7 cells. Bcells. Cells were transiently transfected with CRE reporter plasmid or GRE-reporter plasmdescribed in figure legend of Fig. 1b. Data represent mean ± SD with 6 different samples (and GR from nuclear fractions, and phosphorylated-CREB (p-CREB) from total cell lysates(upper) and aromatase reporter activity (lower) in MCF-7 and TAMR-MCF-7 cells. MCF-7well). Data represent mean ± SD with 4 different samples (significant versus control siR


two cell types (Fig. 2B). Western blotting confirmed that the levelsof Ser133-phosphorylated active CREB and nuclear CREB wereincreased in TAMR-MCF-7 cells compared to MCF-7 cells, whileGR expression was not changed (Fig. 2C). Moreover, CREB knock-down by siRNA decreased protein expression of CREB (Fig. 2D,upper) and significantly suppressed the aromatase reporter activ-ity in TAMR-MCF-7 cells (Fig. 2D, lower).

Role of the PTEN/PI3K/Akt pathway in the up-regulation of aromatasein TAMR-MCF-7 cells

The PI3K/Akt, ERK and p38 kinase signaling pathways areinvolved in the regulation of aromatase activity and CREBactivation in breast cancer cells [15,30]. Our previous studies[21,22] determined that the phospho-active forms of these kinaseswere enhanced in TAMR-MCF-7 cells (Fig. 3A). However, blockingof ERK with PD98059 or p38 kinase with SB203580 did not affectaromatase expression in TAMR-MCF-7 cells (Fig. 3B). On the con-trary, PI3K inhibition by LY 294002 suppressed the enhancedexpression of aromatase in TAMR-MCF-7 cells (Fig. 3B). To confirm

asal activities of minimal reporter of (A) CRE and (B) GRE in MCF-7 and TAMR-MCF-7id (1 lg/ml) and phRL-SV (hRenilla) (1 ng/ml). Reporter activity was determined assignificant versus MCF-7 cells, ��P < 0.01). (C) Immunoblot analyses of nuclear CREBin MCF-7 and TAMR-MCF-7 cells. (D) Effect of CREB siRNA on protein levels of CREBcell and TAMR-MCF-7 cells were transfected with control or CREB siRNA (30 pmol/

NA-transfected TAMR-MCF-7 cells, ��P < 0.01).



241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

Fig. 3. Role of PI3K/Akt signaling pathway in the up-regulation of aromatase expression in TAMR-MCF-7 cells. (A) Immunoblot analyses of p-Akt, Akt, p-ERK, ERK, p-p38kinase and p38 kinase in MCF-7 and TAMR-MCF-7 cells. (B) Effect of kinase inhibitors on aromatase expression in TAMR-MCF-7 cells. TAMR-MCF-7 cells were incubated withPD98059 (PD, 20 lM), SB203580 (SB, 20 lM) and LY294002 (LY, 20 lM) for 24 h and total cell lysates were subjected to immunoblotting. (C), left: effect of p85-myc(dominant negative mutant form of PI3K) transfection on aromatase-luc reporter activity in TAMR-MCF-7 cells. TAMR-MCF-7 cells were transiently co-transfected witharomatase-luc (1 lg/ml), and pCMV5 or p85-myc (0.5 lg/ml) plasmids. Dual luciferase reporter assays were performed as described in the figure legend of Fig. 1b. Datarepresent mean ± SD with 4 different samples (significant versus MCF-7 cells, ��P < 0.01; and significant versus control pCMV5-transfected TAMR-MCF-7 cells ##P < 0.01). (C),right: effect of p110-myc (active form of PI3K) transfection on aromatase-luc reporter activity in MCF-7 cells. MCF-7 cells were transiently co-transfected with aromatase-luc(0.5 lg/ml), and pCMV5 or p110-myc (0.2 lg/ml) plasmids. Dual luciferase reporter assays were performed as described in the figure legend of Fig. 1b. Data representmean ± SD with 3 different samples (significant versus control pCMV5-transfected MCF-7 cells, ��P < 0.01). (D) Effects of PTEN overexpression on protein expression and genetranscription of aromatase in TAMR-MCF-7 cells. TAMR-MCF-7 cells were transfected with PTEN overexpression vector (0.5 lg/ml) and protein expression of aromatase (left)and aromatase promoter activity (right) were determined. Data represent mean ± SD with 4 different samples (significant versus pCMV5-transfected MCF-7 cells, ��P < 0.01;significant versus pCMV5-transfected TAMR-MCF-7 cells, ##P < 0.01). (E) Effects of 5-aza-20-deoxycytidine (5-Aza, DNA methylation inhibitor) on PTEN expression, AKTphosphorylation and aromatase expression in TAMR-MCF-7 cells. TAMR-MCF-7 cells were incubated with 30 lM 5-Aza for 24 h. (F) Effects of LY294002 and 5-Aza on thearomatase activity in TAMR-MCF-7 cells. TAMR-MCF-7 cells were incubated with 20 lM LY294002 or 30 lM 5-Aza for 24 h. Data represent mean ± SD with 3 differentsamples (significant versus vehicle-treated TAMR-MCF-7 cells, ��P < 0.01).



19 May 2014

the role of PI3K in transactivation of the aromatase gene, TAMR-MCF-7 cells was co-transfected with a dominant negative form ofPI3K (p85-myc) and aromatase-luc reporter activity was assessed.The increased promoter activity was significantly reversed by myc-p85 overexpression (Fig. 3C, left). Moreover, co-transfection withconstitutive active form of PI3K (p110-myc) significantly enhancedthe aromatase-luc reporter activity by about 4-fold in MCF-7 cells(Fig. 3C, right).

Phosphatase and tensin homolog (PTEN) is an upstream antag-onistic phosphatase of PI3K [31–33]. We reported previously thatthe loss of PTEN expression via promoter methylation coincidedwith constitutive activation of PI3K in TAMR-MCF-7 cells [21].Thus, we hypothesized that a recovery of PTEN activity in TAMR-MCF-7 cells might inhibit the elevated expression of aromatase.When TAMR-MCF-7 cells were transfected with a plasmid express-ing PTEN, basal expression of PTEN protein was recovered and aro-matase expression concomitantly decreased in TAMR-MCF-7 cells


(Fig. 3D, left). In addition, aromatase promoter reporter activitywas reduced by PTEN expression (Fig. 3D, right). The DNA methyl-ation inhibitor 5-aza-20-deoxycytidine (5-Aza), which reversesPTEN down-regulation and PI3K/Akt activation in TAMR-MCF-7cells by targeting aberrant hypermethylation in the PTEN promoter[21], also significantly reduced aromatase expression in TAMR-MCF-7 cells (Fig. 3E). Moreover, LY294002 or 5-Aza treatmentsinhibited aromatase activity, as measured by a 3[H] water releaseassay (Fig. 3F). These data confirm that deregulation of the PTEN/PI3K/Akt pathway plays a crucial role in up-regulation of aroma-tase expression and activity in TAM-resistant breast cancer cells.

PI3K/Akt-dependent CREB activation in TAMR-MCF-7 cells

Because both CREB activation and PI3K/Akt signaling arerequired for aromatase up-regulation in TAMR-MCF-7 cells, weassessed whether the PI3K/Akt pathway mediates CREB activity.



273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

Fig. 3 (continued)



19 May 2014

Western blotting showed that LY294002 completely inhibited thephosphorylation and nuclear accumulation of CREB in TAMR-MCF-7 cells (Fig. 4A). Minimal CRE reporter activity was also sup-pressed by either LY294002 treatment or co-transfection with thedominant negative PI3K in TAMR-MCF-7 cells (Fig. 4B and C). Thissuggests that PI3K/Akt is involved in CREB activation by stimulat-ing recruitment of CREB from the cytoplasm into the nucleus,which could be responsible for transactivation of the aromatasegene. We further showed that PTEN overexpression (Fig. 4D) or5-Aza treatment (Fig. 4E) significantly reduced CRE reporter activ-ity. In addition, CREB phosphorylation and nuclear accumulation ofCREB in TAMR-MCF-7 cells was diminished by 5-Aza treatment(Fig. 4F). Our findings imply that PI3K/Akt-dependent CREB activa-tion is required for aromatase up-regulation in TAM-resistantbreast cancer.

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

Discussion

Aromatase expression and estrogen levels are frequently ele-vated in breast cancer tissue compared to normal mammary tissue[11,34–37]. Up-regulation of aromatase is believed to stimulatebreast cancer growth in either an autocrine or paracrine manner[38]. It has been also suggested that aromatase can be convertedinto a more active form in growth factor(s)-exposed breast cancercells via post-transcriptional modification [15]. Our current studyfocused on the regulation of aromatase expression in TAM-resis-tant breast cancer. Both the transcriptional activity and proteinexpression of aromatase were enhanced in TAMR-MCF-7 cellscompared to MCF-7 cells. Aromatase expression was also up-regu-lated in a different tamoxifen-resistant breast cancer cell line,T47D:A18/PKCa cells which is stably overexpressing PKCa and


ER function is down-regulated [26]. Moreover, aromatase immu-noreactivity in cancer tissues was increased in recurrence groupafter TAM therapy compared to non-recurrence group after TAMtherapy. From these results, we can conclude that aromataseexpression and its increased activity is one typical feature correlat-ing with the acquisition of TAM resistance. Our finding supports arationale and a mechanistic basic for the acquired benefits of aro-matase inhibitors in relapse prevention of TAM-resistant breastcancer in the clinic [17,18].

The regulation of aromatase expression is dependent on multi-ple tissue-specific promoters [13,14]. In breast cancer tissue, aro-matase transcription is primarily regulated by cAMP-stimulatedpromoters 1.3 [37] and II [36], while in normal mammary tissueit is driven by the glucocorticoid-stimulated promoter 1.4 [39].CRE1 and CRE2 are essential for cAMP-induced promoter II activity[40]. CREB can be phosphorylation by cAMP-dependent proteinkinase at Ser-133 [41]. The activated CREB binds to the CRE pro-moter region and initiates transcription in response to a varietyof extracellular signals, including hormones and growth factors[42]. Here, we have shown that CREB, but not GR, is responsiblefor enhanced aromatase transactivation in TAMR-MCF-7 cells.

Several kinase families play a key role in the activity of mosttranscription factors in response to diverse stimuli [43]. Ser-133phosphorylation of CREB is under the control of ERK and p38kinase [44–46]. In our previous study, ERK, but not p38 kinase,was associated with CREB-mediated aromatase overexpression inp53-inactivated mammary epithelial cells [30]. Although bothkinases were consistently activated in TAMR-MCF-7 cells, inhibi-tion of ERK or p38 kinase did not affect expression of aromatase,demonstrating that the MAPK family is not required for up-regula-tion of aromatase in these cells. Instead, we found that both theexpression and activity of aromatase and CREB were suppressed



334

335

336

337

338

339

340

341

342

343

344

345

346

347

Fig. 4. PI3K/Akt-dependent CREB activation in TAMR-MCF-7 cells. (A) Effect of PI3K inhibition on CREB activity. TAMR-MCF-7 cells were incubated with 20 lM LY294002 for24 h and nuclear CREB and phosphorylated-CREB (p-CREB) were determined. (B), (C) Effect of PI3K inhibition on CRE minimal reporter activity in TAMR-MCF-7 cells. TAMR-MCF-7 cells were incubated with LY294002 (10, 20 lM, B) or transfected with p85-myc (0.5 lg/ml, C) and minimal CRE reporter activity was determined as described in thefigure legend of Fig. 1b. Data represent mean ± SD with 3 different samples (significant versus MCF-7 cells, ��P < 0.01; significant versus vehicle-treated or PCMV5-transfectedTAMR-MCF-7 cells, ##P < 0.01. (D) Inhibition of CRE reporter activity by PTEN overexpression. TAMR-MCF-7 cells were transfected with PTEN overexpression vector (0.5 lg/ml) and minimal CRE reporter activity was determined as described in the legend of Fig. 1b. Data represent mean ± SD with 3 different samples (significant versus pCMV5-transfected MCF-7 cells, ��P < 0.01; significant versus PCMV5-transfected TAMR-MCF-7 cells, ##P < 0.01). (E), (F) Effects of 5-Aza on CREB activation in TAMR-MCF-7 cells.TAMR-MCF-7 cells were incubated with 5-Aza (30 lM) for 24 h and minimal CRE reporter activity (E), and nuclear CREB and phosphorylated-CREB (p-CREB) (F) weredetermined in TAMR-MCF-7 cells. Dual luciferase reporter assays were performed as described in the legend of Fig. 1b. Data represent mean ± SD with 3 different samples(significant versus vehicle-treated MCF-7 cells, ��P < 0.01; significant versus vehicle-treated TAMR-MCF-7 cells, ##P < 0.01).



19 May 2014

by PI3K inhibition, suggesting a role for PI3K signaling in CREB-dependent aromatase expression in TAMR-MCF-7 cells. In fact, sev-eral previous studies have reported the role of PI3K in regulatingaromatase expression in breast cancers. For instance, the anti-estrogen ICI-182,780 suppressed aromatase activity in MCF-7 cellsoverexpressing aromatase by decreasing Akt phosphorylation [47].Another study showed that aromatase activity in breast cancer


cells was controlled by growth factor signaling pathways, includ-ing PI3K/Akt [15]. These reports support our finding that PI3K playsa pivotal role in aromatase up-regulation in TAMR-MCF-7 cells.

PI3K activity is required for cell growth and the acquiredchemo-resistance of breast cancers [48,49], and its activity is neg-atively regulated by PTEN expression [31–33]. We have demon-strated previously that DNA promoter methylation is a key



348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385386387388389390391392393394395396397398399400401402403404405406407408409410411412413

414415416417418419420421422423424425426427428429430431432433



19 May 2014

mechanism mediating the loss of PTEN expression, as well asabnormal proliferation and chemo-resistance in TAMR-MCF-7 cells[21]. 5-Aza, a DNA methylation inhibitor, restored the loss of PTENexpression and inhibited enhanced activation of the PI3K/Akt path-way, consequently recovering endocrine resistance of TAM-resis-tant breast cancer [21]. Thus, down-regulation of aromataseexpression by 5-Aza or PTEN overexpression in the present studyconfirms a close correlation between PTEN/PI3K/Akt signalingand aromatase expression in TAM-resistant breast cancer.

In conclusion, we have shown that aromatase expression is up-regulated through PI3K/Akt-dependent CREB activation in TAM-resistant breast cancer. We further suggest that chemo-resistanceto TAM is partially related to the induction of aromatase, whichprovides a rationale for aromatase targeted therapy in TAM-resis-tant human breast cancer.

434435436437438439440441442443444445446447448449450451452453454455456457458459460461462463464465466467468469470471472473474475476477478479480481482483484485486487488489490491492493494495496497498499

Conflict of Interest

We wish to confirm that there are no known conflicts of interestassociated with this publication and there has been no significantfinancial support for this work that could have influenced itsoutcome.

We confirm that the manuscript has been read and approved byall named authors and that there are no other persons who satis-fied the criteria for authorship but are not listed. We furtherconfirm that the order of authors listed in the manuscript has beenapproved by all of us.

We confirm that we have given due consideration to the protec-tion of intellectual property associated with this work and thatthere are no impediments to publication, including the timing ofpublication, with respect to intellectual property. In so doing weconfirm that we have followed the regulations of our institutionsconcerning intellectual property.

Acknowledgements

This work was supported by the National Research Foundationof Korea (NRF) grant funded by the Korea government (MSIP) (Nos.2007-0056817 and 2011-0029091). All authors declare no conflictsof interest.

References

[1] S.O. Mueller, J.A. Clark, P.H. Myers, K.S. Korach, Mammary gland developmentin adult mice requires epithelial and stromal estrogen receptor alpha,Endocrinology 143 (2002) 2357–2365.

[2] E. Petrangeli, C. Lubrano, F. Ortolani, L. Ravenna, A. Vacca, S. Sciacchitano, L.Frati, A. Gulino, Estrogen receptors: new perspectives in breast cancermanagement, J. Steroid Biochem. Mol. Biol. 49 (1994) 327–331.

[3] J. Pasqualini, G. Chetrite, C. Blacker, M. Feinstein, L. Delalonde, M. Talbi, C.Maloche, Concentrations of estrone, estradiol, and estrone sulfate andevaluation of sulfatase and aromatase activities in pre-and postmenopausalbreast cancer patients, J. Clin. Endocrinol. Metab. 81 (1996) 1460–1464.

[4] S. Ali, R.C. Coombes, Endocrine-responsive breast cancer and strategies forcombating resistance, Nat. Rev. Cancer 2 (2002) 101–112.

[5] C.K. Osborne, S.A. Fuqua, Mechanisms of tamoxifen resistance, Breast CancerRes. Treat. 32 (1994) 49–55.

[6] K. Toda, Y. Shizuta, Molecular cloning of a cDNA showing alternative splicing ofthe 50-untranslated sequence of mRNA for human aromatase P-450, Eur. J.Biochem. 213 (1993) 383–389.

[7] D. Ghosh, J. Griswold, M. Erman, W. Pangborn, Structural basis for androgenspecificity and oestrogen synthesis in human aromatase, Nature 457 (2009)219–223.

[8] D. Köberle, B. Thürlimann, Adjuvant endocrine therapy in postmenopausalbreast cancer patients, Breast 14 (2005) 446–451.

[9] A.J. Wood, I.E. Smith, M. Dowsett, Aromatase inhibitors in breast cancer, N.Engl. J. Med. 348 (2003) 2431–2442.

[10] R.W. Brueggemeier, J.C. Hackett, E.S. Diaz-Cruz, Aromatase inhibitors in thetreatment of breast cancer, Endocr. Rev. 26 (2005) 331–345.

[11] S. Chen, Aromatase and breast cancer, Front Biosci. 3 (1998) d922–d933.[12] N. Harada, Aberrant expression of aromatase in breast cancer tissues, J. Steroid

Biochem. Mol. Biol. 61 (1997) 175–184.


[13] G.D. Means, M.W. Kilgore, M.S. Mahendroo, C.R. Mendelson, E.R. Simpson,Tissue-specific promoters regulate aromatase cytochrome P450 geneexpression in human ovary and fetal tissues, Mol. Endocrinol. 5 (1991)2005–2013.

[14] T. Utsumi, N. Harada, M. Maruta, Y. Takagi, Presence of alternatively splicedtranscripts of aromatase gene in human breast cancer, J. Clin. Endocrinol.Metab. 81 (1996) 2344–2349.

[15] B. Su, C. Wong, Y. Hong, S. Chen, Growth factor signaling enhances aromataseactivity of breast cancer cells via post-transcriptional mechanisms, J. SteroidBiochem. Mol. Biol. 123 (2011) 101–108.

[16] S. Chumsri, T. Howes, T. Bao, G. Sabnis, A. Brodie, Aromatase, aromataseinhibitors, and breast cancer, J. Steroid Biochem. Mol. Biol. 125 (2011) 13–22.

[17] M. Kaufmann, W. Jonat, J. Hilfrich, H. Eidtmann, G. Gademann, I. Zuna, G. vonMinckwitz, Improved overall survival in postmenopausal women with earlybreast cancer after anastrozole initiated after treatment with tamoxifencompared with continued tamoxifen: the ARNO 95 Study, J. Clin. Oncol. 25(2007) 2664–2670.

[18] F. Boccardo, A. Rubagotti, P. Guglielmini, A. Fini, G. Paladini, M. Mesiti, M.Rinaldini, S. Scali, M. Porpiglia, C. Benedetto, N. Restuccia, F. Buzzi, R. Franchi,B. Massidda, V. Distante, D. Amadori, P. Sismondi, Switching to anastrozoleversus continued tamoxifen treatment of early breast cancer. Updated resultsof the Italian tamoxifen anastrozole (ITA) trial, Ann. Oncol.: Official J. Eur. Soc.Med. Oncol./ESMO, 17 Suppl 7 (2006) vii 10–14.

[19] M.K. Cho, S.G. Kim, Hepatocyte growth factor activates CCAAT enhancerbinding protein and cell replication via PI3-kinase pathway, Hepatology 37(2003) 686–695.

[20] H.K. Choi, J.W. Yang, S.H. Roh, C.Y. Han, K.W. Kang, Induction of multidrugresistance associated protein 2 in tamoxifen-resistant breast cancer cells,Endocr. Relat. Cancer 14 (2007) 293–303.

[21] N.T.T. Phuong, S.K. Kim, S.C. Lim, H.S. Kim, T.H. Kim, K.Y. Lee, S.-G. Ahn, J.-H.Yoon, K.W. Kang, Role of PTEN promoter methylation in tamoxifen-resistantbreast cancer cells, Breast Cancer Res. Treat. 130 (2011) 73–83.

[22] S.K. Kim, J.W. Yang, M.R. Kim, S.H. Roh, H.G. Kim, K.Y. Lee, H.G. Jeong, K.W.Kang, Increased expression of Nrf2/ARE-dependent anti-oxidant proteins intamoxifen-resistant breast cancer cells, Free Radical Biol. Med. 45 (2008) 537–546.

[23] Y. Kinoshita, S. Chen, Induction of aromatase (CYP19) expression in breastcancer cells through a nongenomic action of estrogen receptor a, Cancer Res.63 (2003) 3546–3555.

[24] M. Ito, J.C. Achermann, J.L. Jameson, A naturally occurring steroidogenic factor-1 mutation exhibits differential binding and activation of target genes, J. Biol.Chem. 275 (2000) 31708–31714.

[25] X. Wang, X. Zhao, X. Gao, Y. Mei, M. Wu, A new role of p53 in regulating lipidmetabolism, J. Mol. Cell Biol. 5 (2013) 147–150.

[26] D. Tonetti, M. Chisamore, W. Grdina, H. Schurz, V. Jordan, Stable transfection ofprotein kinase C alpha cDNA in hormone-dependent breast cancer cell lines,Br. J. Cancer 83 (2000) 782.

[27] E.R. Simpson, Y. Zhao, Estrogen biosynthesis in adipose, Ann. N. Y. Acad. Sci.784 (1996) 18–26.

[28] M. Young, M.J. McPhaul, A steroidogenic factor-1-binding site and cyclicadenosine 30 , 50-monophosphate response element-like elements are requiredfor the activity of the rat aromatase promoter in rat Leydig tumor cell lines,Endocrinology 139 (1998) 5082–5093.

[29] S. Chen, D. Zhou, C. Yang, T. Okubo, Y. Kinoshita, B. Yu, Y.-C. Kao, T. Itoh,Modulation of aromatase expression in human breast tissue, J. SteroidBiochem. Mol. Biol. 79 (2001) 35–40.

[30] H.K. Choi, S.H. Roh, H.G. Kim, E.H. Han, H.G. Jeong, K.W. Kang, Enhancedexpression of aromatase in p53-inactivated mammary epithelial cells, Endocr.Relat. Cancer 15 (2008) 139–147.

[31] J.Á. Fresno Vara, E. Casado, J. de Castro, P. Cejas, C. Belda-Iniesta, M. González-Barón, PI3K/Akt signalling pathway and cancer, Cancer Treat. Rev. 30 (2004)193–204.

[32] Y. Samuels, K. Ericson, Oncogenic PI3K and its role in cancer, Curr. Opin. Oncol.18 (2006) 77–82.

[33] V. Stambolic, A. Suzuki, J.L. De La Pompa, G.M. Brothers, C. Mirtsos, T. Sasaki, J.Ruland, J.M. Penninger, D.P. Siderovski, T.W. Mak, Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN, Cell 95 (1998) 29–39.

[34] V. James, J. McNeill, L. Lai, C. Newton, M. Ghilchik, M. Reed, Aromatase activityin normal breast and breast tumor tissues: In vivo and in vitro studies, Steroids50 (1987) 269–279.

[35] W. Miller, P. Mullen, P. Sourdaine, C. Watson, J. Dixon, J. Telford, Regulation ofaromatase activity within the breast, J. Steroid Biochem. Mol. Biol. 61 (1997)193–202.

[36] C. Zhou, D. Zhou, J. Esteban, J. Murai, P.K. Siiteri, S. Wilczynski, S. Chen,Aromatase gene expression and its exon I usage in human breast tumors.Detection of aromatase messenger RNA by reverse transcription-polymerasechain reaction, J. Steroid Biochem. Mol. Biol. 59 (1996) 163–171.

[37] D. Zhou, S. Chen, Identification and characterization of a cAMP-responsiveelement in the region upstream from promoter 1.3 of the human aromatasegene, Arch. Biochem. Biophys. 371 (1999) 179–190.

[38] X.-Z. Sun, D. Zhou, S. Chen, Autocrine and paracrine actions of breast tumoraromatase. A three-dimensional cell culture study involving aromatasetransfected MCF-7 and T-47D cells, J. Steroid Biochem. Mol. Biol. 63 (1997)29–36.



500501502503504505506507508509510511512513514515516

517518519520521522523524525526527528529530531532533



19 May 2014

[39] S. Chen, T. Itoh, K. Wu, D. Zhou, C. Yang, Transcriptional regulation ofaromatase expression in human breast tissue, J. Steroid Biochem. Mol. Biol. 83(2002) 93–99.

[40] M. Sofi, M.J. Young, T. Papamakarios, E.R. Simpson, C.D. Clyne, Role of CRE-binding protein (CREB) in aromatase expression in breast adipose, BreastCancer Res. Treat. 79 (2003) 399–407.

[41] M. Johannessen, M.P. Delghandi, U. Moens, What turns CREB on?, Cell Signal.16 (2004) 1211–1227.

[42] P.J. Deutsch, J. Hoeffler, J.L. Jameson, J. Lin, J.F. Habener, Structuraldeterminants for transcriptional activation by cAMP-responsive DNAelements, J. Biol. Chem. 263 (1988) 18466–18472.

[43] R. Treisman, Regulation of transcription by MAP kinase cascades, Curr. Opin.Cell Biol. 8 (1996) 205–215.

[44] M. Cammarota, L.R.M. Bevilaqua, P.R. Dunkley, J.A.P. Rostas, Angiotensin IIpromotes the phosphorylation of cyclic AMP-responsive element bindingprotein (CREB) at Ser133 through an ERK1/2-dependent mechanism, J.Neurochem. 79 (2001) 1122–1128.

534


[45] R. Hokari, H. Lee, S.C. Crawley, S.C. Yang, J.R. Gum, S. Miura, Y.S. Kim,Vasoactive intestinal peptide upregulates MUC2 intestinal mucin via CREB/ATF1, Am. J. Physiol. – Gastrointestinal Liver, Physiol. 289 (2005) G949–G959.

[46] D.P. Gelain, M. Cammarota, A. Zanotto-Filho, R.B. de Oliveira, F. Dal-Pizzol, I.Izquierdo, L.R. Bevilaqua, J.C. Moreira, Retinol induces the ERK1/2-dependentphosphorylation of CREB through a pathway involving the generation of reactiveoxygen species in cultured Sertoli cells, Cell. Signal. 18 (2006) 1685–1694.

[47] G. Sabnis, O. Goloubeva, D. Jelovac, A. Schayowitz, A. Brodie, Inhibition of thephosphatidylinositol 3-kinase/Akt pathway improves response of long-termestrogen-deprived breast cancer xenografts to antiestrogens, Clin. Cancer Res.13 (2007) 2751–2757.

[48] T. Frogne, J. Jepsen, S. Larsen, C. Fog, B. Brockdorff, A. Lykkesfeldt, Antiestrogen-resistant human breast cancer cells require activated protein kinase B/Akt forgrowth, Endocr. Relat. Cancer 12 (2005) 599–614.

[49] N.J. Jordan, J.M. Gee, D. Barrow, A.E. Wakeling, R.I. Nicholson, Increasedconstitutive activity of PKB/Akt in tamoxifen resistant breast cancer MCF-7cells, Breast Cancer Res. Treat. 87 (2004) 167–180.



Aromatase induction in tamoxifen-resistant breast cancer: Role of phosphoinositide...

Documents

Transcript of Aromatase induction in tamoxifen-resistant breast cancer: Role of phosphoinositide...